Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007

Abstract

Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit.

Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

“…An important drawback in the application of MCMOs has been the requirement for a FR-type protein that can serve as a robust effective shuttle for the requisite reduced FMN. During our studies on the recombinant production of both DKCMOs from NCIMB 10007 (Kadow et al 2011; we discovered that an enzyme activity in E. coli was apparently fulfilling this key shuttle role for these Type II BVMOs.…”

“…Expression of 2,5-and 3,6-DKCMO was performed as described elsewhere (Kadow et al 2011;. Coexpression of Fre together with a DKCMO was achieved in terrific broth (TB) medium supplemented with kanamycin and carbenicillin plus chloramphenicol in case of coexpression of chaperones GroES/GroEL.…”

“…The relevant isoenzymes are 2,5-and 3,6-diketocamphane monooxygenase (2, that catalyse the biooxidation of the diketocamphane antipodes derived from (+)-or (-)-camphor to the corresponding lactones, which subsequently undergo spontaneous ring opening thereby triggering further catabolic dissimilation (Conrad et al 1965a;Jones et al 1993;Taylor and Trudgill 1986). The two DKCMOs were recently cloned and recombinantly expressed (Kadow et al 2011; but their exceptional catalytic potential was acknowledged much earlier in a number of studies applying whole cells or partly purified fractions obtained from the NCIMB 10007 strain (Gagnon et al 1994;1995;Grogan et al 1993). Both isoenzymes operate in situ as loosely associated trimeric assemblages composed of the biooxygenating component plus a single flavin reductase (FR) (Jones et al 1993).…”

“…Firstly, it has long been recognised that both trimeric assemblages are unstable (Conrad et al 1965a;Taylor and Trudgill 1986) and preferentially use artificial electron acceptors such as 2,6-dichlorophenolindophenol (DCPIP) as effective sinks for transferred reducing power (Gunsalus et al 1965); secondly, the equivalent purified FR components isolated from various MCMO luciferases sourced from bioluminescent bacteria are effective entities in promoting reducing power transfer to the biooxygenating subunits of both DKCMOs (Beecher 1997 (Williams et al 1989) and the luciferase from Vibrio harveyi (Campbell and Baldwin 2009). Recently, two significant additional developments challenged further the long-standing proposed functional relationship of the trimeric DKCMOs first proposed by Gunsalus (Conrad et al 1965a;Gunsalus et al 1965) Initially, it was demonstrated that the genes encoding the biooxygenating subunits of both 2,5-and 3,6-DKCMO were functional when expressed in E. coli (Kadow et al 2011;. Subsequently, it has been shown that the biooxygenating components of each DKCMO isoenzyme perform efficient lactone-forming biooxygenation reactions in association with a homodimeric FR (2 x 18 kDa) encoded by the chromosomal DNA of NCIMB 10007 when tandem plasmid constructs carrying both relevant genes were expressed in E. coli (Iwaki et al 2013).…”

“…In the light of these recent observations, the aim of the work reported here was: (i) to identify the FR enzyme(s) of E. coli responsible for the recently observed supply of reduced FMN to recombinant two-component BVMOs (Kadow et al 2011; and (ii) to assemble any such competent functionality with the biooxygenating components of 2,5-and 3,6-DKCMO to promote their biooxygenating activity both via coexpression of the relevant genes (2,5-DKCMO x FR and 3,6-DKCMO x FR), and more ambitiously, via fusion of the relevant proteins into single open reading frame constructs (2,5-DKCMO-FR and 3,6-DKCMO-FR).…”